We have reported that histone acetylation induced by trichostatin A (TSA) promotes p21 waf1/cip1 (p21) expression, the GC-box located just upstream of TATA box was responsible for TSA-induced promoter activation, and both Sp1 and Sp3 were the working activator of this GC-box. To understand the molecular pathway from histone acetylation to this Sp1 family factors-mediated promoter activation, we investigated the function of p300, one of the histone acetyltransferase, in the present work. The

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... ence supporting the linkage between p300 and TSA-induced p21 promoter activation were realized from the following findings: 1) cotransfection of p300 elevated p21 promoter activity, and this elevation was dependent on TSA-responsive GC-box; 2) TSA-induced promoter activation was blocked by the introduction of p300 dominant-negative mutant into cells; and 3) Sp1-or Sp3-mediated activation was also suppressed by this p300 dominant-negative mutant. Our data also suggested that p300 collaborates with Sp1 in a way which is different from that when p300 collaborates with p53 in p21 transcription. p21 waf1/cip1 (p21) 1 is a gene functioning as a cell cycle blocker, and its expression is usually regulated at transcription level. p21 was first cloned and characterized as an important effector that acts to inhibit cyclin-dependent kinase activity in p53mediated cell cycle arrest induced by DNA damage (1-4). Further studies indicated that p21 is also regulated by other transcription factors during cell differentiation and growth arrest (5-8). During the study of cellular senescence, we found that the inhibitors of histone deacetylase, either sodium butyrate or TSA, can promote p21 transcription and induce growth arrest and senescence-like state in NIH 3T3 cells (9). 2 The minimal region of the mouse p21 promoter, containing from Ϫ60 to ϩ40 bp relative to the TATA box, is essential and sufficient for the induction of p21 promoter by TSA. We reported also that a GC-box in this region is critical for both basal and TSA-induced promoter activity and that Sp1 and Sp3 are the functional activators of this GC-box (10). However, one question re-mained, how does histone acetylation affect Sp1-mediated transcription? Recently, the study of transcriptional regulation has been moving its focus to chromatin level. The molecules involved in chromatin transcription include DNA (promoter, enhancer, or silencer), histones, and non-histone proteins. It has become increasingly apparent that the equilibrium of histone acetylation and deacetylation plays an important role in transcriptional regulation (11, 12) . Several mammalian histone acetyltransferases and histone deacetylases have been cloned in recent years (13) (14) (15) (16) (17) (18) (19) . p300 was first cloned as an E1A associated protein with properties of a transcriptional adapter (20). This protein was found later to possess intrinsic histone acetyltransferase activity and works as a coactivator in MyoD-, p53-, and SRC-1-mediated transcription (21-23). Indirect evidence has implicated p300 in cell cycle control and differentiation (24, 25) . Although p300 has been found to be required for induction of p21 expression in keratinocyte differentiation (26), the ciselement in the p21 promoter and the sequence-specific transcriptional activator remained unknown. The present work is part of our effort to understand the linkage between histone acetylation and Sp1-mediated transcription. Here we show evidence that p300 is required for TSA-induced, Sp1-mediated p21 transcription and discuss the possible mechanism of the functional interaction between Sp1 and p300 in p21 transcriptional activation. EXPERIMENTAL PROCEDURES Cells and Reagents-HeLa cells from ATCC and COS-1 cells from RIKEN (Wako, Japan) were cultured in a 37°C humidified atmosphere containing 5% CO 2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Trichostatin A (Wako, Osaka) was dissolved with ethanol at a concentration of 1 mg/ml and stored at Ϫ20°C. The working solution was diluted with distilled phosphatebuffered saline to 10 g/ml concentration and stored at 4°C for less than 1 week. Plasmid Constructs-The full-length (pGL3b-4542), the wild type minimal region (pGL3b-60 or pRL-60), and the GC-box mutated minimal region (pGL3b-#4 or pRL-#4